Fluorescence microscopy plays a major role as a diagnostic tool in many scientific disciplines. The fundamental principle of fluorescence microscopy is that a sample is irradiated with short-wavelength excitation radiation, and that the sample itself, or a fluorescing dye with which the sample is stained, emits longer-wavelength fluorescence light (primary or secondary fluorescence) upon excitation with the short-wavelength excitation wavelength. For fluorescence microscopy, secondary fluorescence is utilized as a rule in order to make specific specimen structures of stained prepared specimens visible. It is thereby possible, for example, to identify pathogens, localize genes, identify genetic changes in a DNA being investigated, or even visualize protein formations in cells.
A particular investigation method using specific fluorescing dyes (called “fluorochromes”) is available depending on the application. Typical excitations, for example, involve UV light for the “DAPI” dye, blue light for the “FITC” dye, or green light for the “Texas Red” or “Rhodamine” dyes. Typical excitation frequencies are in the ultraviolet and visible spectral region.
Conventionally, short-arc lamps filled with Hg or Xe, or halogen lamps, have usually be used as light sources. The spectral region appropriate for exciting a fluorochrome can be selected out of the spectral region of the light source using a variety of (exchangeable) dielectric filters called “excitation filters.” The aforesaid lamps are nowadays being displaced by light-emitting diodes (LEDs) as light sources for fluorescence microscopy.
DE 20 2004 010 121 U1, for example, discloses a light source for an incident-light fluorescence microscope which comprises a high-output LED that emits blue light in a spectral region from 460 to 480 nm.
EP 1 592 996 A2 discloses a system having two light-emitting diodes whose illumination beam paths are combined by a dichroic splitter and directed onto a fluorescence filter system.
Different fluorescence filter systems, also referred to as “filter blocks” or “filter cubes,” are typically worked with so that different stains of a prepared specimen can be visualized. These fluorescence filter systems have hitherto been made up of a mutually coordinated combination of an excitation filter, a dichroic splitter, and a blocking filter. The dichroic splitter reflects toward the prepared specimen the excitation radiation that the excitation filter allows to pass. The dichroic splitter is, however, transparent to the fluorescence light emitted from the prepared specimen. The blocking filter holds back excitation light that is scattered from the prepared specimen and enters the objective. It possesses maximum transparency, however, for the specific fluorescence radiation given off by the specimen.
The various fluorescence filter systems are usually located on a changing device that is embodied, for example, as a slider or carousel. Operation is effected in manual and/or motorized fashion.
DE 10 2007 007 797 A1 relates to a fluorescence microscope having an incident fluorescence illumination device. The illumination device encompasses several light-emitting diodes having upstream collectors for generating a directed light flux, the relevant light fluxes being combined by means of dichroic splitters into one common illumination beam path that in turn strikes a multi-band excitation filter of a multi-band fluorescence filter system. The latter encompasses a multi-band beam splitter as well as a multi-band blocking filter. The multi-band excitation filter is transparent to the wavelength regions of the respective light-emitting diodes, while the multi-band blocking filter is as opaque as possible to those wavelength regions but transmits the corresponding emission bands of the fluorescence radiation emitted from the specimen. Control is applied to the light-emitting diodes via a common logical control device in order to allow rapid switchover between excitation wavelengths. In the fluorescence microscope described therein, no provision is made for bright-field transmitted-light observation of the specimens.
In addition to incident-light fluorescence microscopy, it is often desirable also to investigate or observe specimen structures using bright-field transmitted-light microscopy. Fluorescence filter changing apparatuses are used to switch over between fluorescence imaging and bright-field imaging; these apparatuses are of complex design and cost-intensive to manufacture, and furthermore are slow to switch over between fluorescence imaging and bright-field imaging.